Reverse Transcription Coupled To Polymerase Chain Reaction Research Articles

Growth hormone-related genes from baboon ( Papio hamadryas): Characterization, placental expression and evolutionary aspects

Pregnancy is a complex physiological condition, and the growth hormone (GH)-related hormones produced in the placenta, which emerged during the evolution of primates, are thought to play an important metabolic role in pregnancy that is not yet fully understood. The aim of this study was to identify the genes and transcription products of the GH family in baboon ( Papio hamadryas) and to assess these in relation to the evolution of this gene family. GH-related transcripts were amplified using total RNA from placental tissue, by reverse transcription coupled to polymerase chain reaction (RT-PCR). Three different GH-related transcripts were identified in baboon placental tissue, with two encoding chorionic somatomammotropins (CSH) and one the placental variant of GH ( GH-2). The CSH transcripts showed some minor allelic variation, and a splice variant of CSH-C that retains its in-frame third intron. Gene sequences for GH-1 (probably representing the GH gene expressed primarily in the pituitary gland), GH-2 and the two CSHs were identified in the baboon genomic database, together with a CSH-related pseudogene. Phylogenetic analysis of the baboon GH-related sequences, together with those of a related Old World monkey, macaque, and ape outgroup (human), showed the equivalence of the genes in baboon and macaque, and revealed evidence for several episodes of rapid adaptive evolution. Many of the substitutions seen during the evolution of these placental proteins have occurred in the receptor-binding sites, especially site 2, contrasting with the strong conservation of the hydrophobic core.

Expression analysis of the AtMLO Gene Family Encoding Plant-Specific Seven-Transmembrane Domain Proteins

The Arabidopsis (Arabidopsis thaliana) genome contains 15 genes encoding protein homologs of the barley mildew resistance locus o (MLO) protein biochemically shown to have a seven-transmembrane domain topology and localize to the plasma membrane. Towards elucidating the functions of MLOs, the largest family of seven-transmembrane domain proteins specific to plants, we comprehensively determined AtMLO gene expression patterns by a combination of experimental and in silico studies. Experimentation comprised analyses of transgenic Arabidopsis lines bearing promoter::Beta-glucuronidase (GUS) transcriptional fusions as well as semi-quantitative determination of transcripts by reverse transcription coupled to polymerase chain reaction (RT-PCR). These results were combined with information extracted from public gene profiling databases, and compared to the expression patterns of genes encoding the heterotrimeric G-protein subunits. We found that each AtMLO gene has a unique expression pattern and is regulated differently by a variety of biotic and/or abiotic stimuli, suggesting that AtMLO proteins function in diverse developmental and response processes. The expression of several phylogenetically closely-related AtMLO genes showed similar or overlapping tissue specificity and analogous responsiveness to external stimuli, suggesting functional redundancy, co-function, or antagonistic function(s).

Endogenous morphinergic signaling and tumor growth.

The mu3 opiate receptor subtype has been characterized by various binding assays as opiate alkaloid selective (e.g. morphine) and opioid peptide (e.g. methionine enkephalin) insensitive. This opiate receptor subtype has been found on human, including cancer cell lines, and invertebrate tissues, demonstrating that it has been conserved during evolution. Furthermore, in numerous reports, this receptor is coupled to constitutive nitric oxide release. In this regard, for example, morphine immune down regulating activities parallels those actions formerly attributed to nitric oxide. We have now identified the mu3 receptor at the molecular level and sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene (MOR). Furthermore, using Northern blot, reverse transcription coupled to polymerase chain reaction (RT-PCR) and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells. The presence of this mu splice variant, adds to the growing body of evidence supporting the hypothesis that morphine is an endogenous signaling molecule in neural, immune and vascular systems. In addition to their use in the treatment of pain, opioid peptides appear to be important in the growth regulation of normal and neoplastic tissue. This review will focus on the influence of opiate alkaloids, e.g., morphine, on tumor growth, with emphasis on immuno-regulatory and antiproliferative mechanisms.

Nested rt-PCR in a single closed tube.

There are several methods now in widespread use for detecting and characterizing specific RNA targets. These methods include in situ hybridization, Northern blotting, dot or slot blot, RNase protection assay, and reverse transcription coupled to polymerase chain reaction (RT-PCR). However, when the amount of RNA target is limited or restricted in its cellular or tissue distribution, the extreme sensitivity of the PCR allows the detection of minute quantities of RNA when coupled to an initial step that converts single-strand RNA to cDNA (, , , ). Nevertheless, when RT-PCR is applied for diagnostic purposes, the sensitivity usually afforded by this technique in routine detection tests is similar, or only slightly higher, to conventional enzyme-linked immunosorbent assay or hybridization techniques. This is frequently observed when dealing with poor-quality samples containing inhibitors of RT-PCR. The presence of different components of plant or animal origin, as well as specific RT-PCR conditions, may inhibit the reverse transcription and amplification by a number of mechanisms ().

Expression and promoter activation of the Rpe65 gene in retinal pigment epithelium cell lines

Purpose. To examine the expression and promoter activation of the retinal pigment epithelium (RPE)-preferentially expressed Rpe65 gene in the commonly available RPE cell lines. Methods. Reverse transcription coupled to polymerase chain reaction (RT-PCR) was performed after total RNA extraction from different RPE (ARPE-19, monkey, hTERT-RP1 and D407) and non-RPE (COS-7, HeLa, HepG2 and HS27) cell lines. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the 5' flanking region of the mouse Rpe65 gene. The involvement of a putative TATA box in the basal promoter expression was studied by site-directed mutagenesis in D407 cells and binding of TATA box-related transcription factors to that region was demonstrated by Electrophoretic Mobility Shift Assays (EMSA). Results. Expression of the human RPE65 cDNA was observed in all the RPE cell lines tested, and in COS-7 cells (monkey RPE65 cDNA). Transient transfections of the mouse Rpe65 promoter/luciferase transgene containing nested deletions of the Rpe65 5' flanking region showed that fragments containing bases -655 to +48 and -1240 to +48 generated specific promoter activity only in the D407 cell line. A promoter fragment from -49 to +48 directed basal promoter activity in all the cell lines tested. Part of this basal activity was due to a putative TATA box that specifically binds transcription factors contained in a D407 nuclear extract. Conclusion. Although transcription of the Rpe65 gene occurs in all the tested cell lines, we find that the D407 cell line is the only one capable of directing specific mouse Rpe65 promoter activity. This limits the study of the transcriptional regulation of the mouse Rpe65 gene in vitro to this particular cell line.

Retinoid X receptor mRNA expression in human pituitary gland.

Retinoic acid is involved in important physiological processes such as the regulation of growth and differentiation of several tissues, including the pituitary gland. These biological effects are mediated by their binding to two specific intracellular receptors termed retinoic acid and retinoid X receptors, RARs, RXR, respectively). RAR or RXR mRNA expression has been demonstrated in several tissues, but little information is available about its presence in the human pituitary gland. In this report, we demonstrate alphaRXR mRNA expression using the reverse transcription coupled to polymerase chain reaction (RT-PCR) in the human pituitary gland. These results suggest the possibility that RXR may regulate the human pituitary gene expression and hormone secretion.

Cloning and Characterization of cDNA Encoding Cdc2 Kinase, a Component of Maturation-Promoting Factor, in Rana dybowskii

In order to understand the mechanism of oocyte maturation in seasonal-breeding wild frogs, we have cloned and sequenced a cDNA encoding Cdc2 kinase, a component of the maturation-promoting factor (MPF) in Rana dybowskii. About 1.2-kb cDNA was isolated by reverse transcription coupled to polymerase chain reaction (RT-PCR) and cDNA library screening. The cloned Rana Cdc2 cDNA encodes a complete open-reading frame with 302 amino acid residues, which deduce a 34-kDa protein. Homology of more than 80% was found between the deduced amino acid sequence of Rana Cdc2 and that of five phylogenetically distant organisms, and 94% identity was found between Rana and Xenopus. More importantly, the Thr14, Tyr15, and Thr161 residues, the phosphorylation sites for the activation of the enzyme, are highly conserved. In vitro-translated Rana Cdc2 cross-reacted with Xenopus p34cdc2 antibody as shown by Western blot. Northern blot analysis showed that a 1.7-kb transcript was highly expressed in the gonads compared to other tissues, indicating the important role of Cdc2 kinase in gonads as a component of MPF. The cloned Rana Cdc2 cDNA also exhibited histone H1 kinase activity when expressed in CV-1 cells. In the present study, therefore, we have characterized the Rana Cdc2 kinase in amphibian, which will be helpful in understanding the process of oocyte maturation related to the reproduction cycle of wild frogs.

Assessment of SNRPN expression as a molecular tool in the diagnosis of Prader-Willi syndrome

Prader-Willi syndrome (PWS) is associated with lesions of the paternal chromosome 15q11- 13. Recently, loss of expression of a paternally expressed gene in this region, SNRPN, has been proposed as a molecular hallmark of PWS. The goal of this study was to determine the diagnostic accuracy of SNRPN expression in a well-characterized cohort of PWS patients. SNRPN expression was analyzed by reverse transcription coupled to polymerase chain reaction (RT-PCR). RNA was isolated from peripheral blood leukocytes and subjected to multiplex RT-PCR in which expression of SNRPN and a constituitively expressed internal control gene were analyzed. The amplified products were electrophoresed in agarose gels and visualized by ethidium bromide staining. Multiplex RT-PCR was applied to RNAs isolated from 30 normal control subjects and 30 well- characterized PWS patients. All control patients expressed the SNRPN and internal control genes. In contrast, all 30 PWS patients demonstrated loss of SNRPN expression, with integrity of RNA being demonstrated by the presence of internal control gene expression. Loss of SNRPN expression appears to be a consistent finding in PWS. Expression analysis of SNRPN offers a novel approach for the diagnostic evaluation of PWS that is robust and can be performed in a single day.

Coexpression of phospholipase A 2 isoforms in rat striatal astrocytes

The expression and activity of phospholipase A 2 (PLA 2) isoforms were investigated in primary cultures of striatal astrocytes. The calcium ionophore A23187 together with the protein kinase C activator phorbol ester was the most potent stimulus in eliciting [ 3H]arachidonic acid release in the extracellular medium. Reverse transcription coupled to polymerase chain reaction (RT-PCR) showed the presence of the 85 kDa cytosolic PLA 2 mRNA and the 14 kDa secretory PLA 2 mRNA in untreated astrocytes. Immunoblot experiments with isoform-specific antibodies showed the presence of the cytosolic PLA 2 in untreated astrocytes, while the secretory PLA 2 was detected only in lipopolysaccharide-treated astrocytes. These data suggest that the two PLA 2 isoforms expressed in striatal astrocytes might play different roles in cellular processes mediated by astrocytes.

Evidence for hormone-sensitive lipase mRNA expression in human monocyte/macrophages.

The role of hormone-sensitive lipase (HSL) in the hydrolysis of adipose tissue triacylglycerol to provide free fatty acids for energy requirements has been well established. However, the role of HSL in other tissues, including macrophages, is not well understood. The demonstration that HSL is capable of hydrolyzing cholesteryl esters at approximately the same rate as triacylglycerol raised the possibility that HSL activity in macrophages may influence the accumulation of cholesteryl esters in foam cells of atherosclerotic lesions. We and others have previously demonstrated that HSL mRNA is expressed in murine peritoneal macrophages and macrophage cell lines; however, it was previously reported that HSL mRNA is absent in human monocyte-derived macrophages, suggesting that a species difference may exist. To clarify this point, we have further examined the issue of HSL mRNA expression in human macrophages. In the current study, we demonstrate that HSL mRNA is detectable in human monocyte-derived macrophages and in the THP-1 human monocyte cell line using reverse transcription coupled to polymerase chain reaction (RT-PCR). Specific amplification of cDNA derived from mRNA was ensured by using primers that span an intron within the human HSL gene, and the identity of PCR products as HSL was confirmed by hybridization to HSL cDNA and by DNA sequencing. Using a semiquantitative PCR assay, we establish that HSL mRNA levels in monocyte/macrophages are approximately 1/40 the levels in human adipose tissue. These results indicate that further studies addressing the role of HSL in macrophage metabolism and its potential role in development of foam cells in human atherosclerotic lesions are warranted.

Localisation of vascular endothelial growth factor and its receptors to cells of vascular and avascular epiretinal membranes

AIMS/BACKGROUNDEpiretinal membranes (ERMs) arise from a variety of causes or, in some cases, for unknown reasons. Once established, ERMs tend to progress, becoming more extensive and exerting increasing traction along...

Expression of progesterone receptor mRNA in the first trimester human placenta.

The human placenta produces large quantities of progesterone, the function and target tissues of action of which during pregnancy are not completely understood. Although it has been suggested that placenta itself could be a potential target tissue for progesterone action, there is no conclusive evidence for the presence of progesterone receptors in the human placenta. We provide evidence for the expression of progesterone receptor mRNA in the human placenta by reverse transcription coupled to polymerase chain reaction (RT-PCR). This was further confirmed by Southern hybridization, restriction analysis and sequencing of the PCR amplified fragment.

Expression of thyroid hormone receptors in human pituitary tumor cells.

To understand the expression of thyroid hormone receptors (TRs) in various types of pituitary tumor cells, we performed the reverse transcription coupled to polymerase chain reaction (RT-PCR). Specimens of pituitary adenomas were obtained by transsphenoidal adenomectomies from 2 patients with acromegaly, 6 patients with prolactinoma, 3 patients with Cushing's disease, 2 patients with gonadotropin-secreting tumors, one patient with a TSH-secreting tumor, and 3 patients with non-functioning adenoma with no clinical evidence of pituitary hormone overproduction. Human thyroid hormone receptors beta 1 and alpha 1, alpha 2 (h-TR beta 1, alpha 1, alpha 2) are expressed in nonfunctioning adenomas; h-TR alpha 1 and beta 1 are specific for prolactinomas; h-TR beta 1 is involved in acromegalies and FSH-secreting tumors, while h-TR alpha 1, alpha 2, and beta 1 are not expressed in TSH-secreting and ACTH-secreting tumors. The results suggest that human thyroid hormone receptors are differentially expressed in human pituitary tumor cells, and our studies have shed new light on the understanding of the role of TRs in pituitary tumors.

17 Beta Hydroxysteroid Dehydrogenase 1 "Pseudogene" Is Differentially Transcribed: Still a Candidate for the Breast-Ovarian Cancer Susceptibility Gene (BRCA1)

BRCA1, the susceptibility gene for hereditary breast-ovarian cancer, is located on chromosome 17q12-21 but has not yet been identified. Two tandem oestradiol 17 beta hydroxysteroid dehydrogenase genes (17HSD) are assigned to this region. The active 17HSDII gene encodes the normal enzyme which regulates local synthesis of oestrogens, whereas 17HSDI is considered to be a pseudogene. We used reverse transcription coupled to polymerase chain reaction (RT-PCR) and found that the 17HSDI gene was also transcribed in half of the human cell lines and most of the biopsies studied, suggesting that 17HSDI could modulate normal 17HSDII activity in oestrogen target cells. We hypothesize that altered 17HSDI gene expression could lead to both hereditary and/or sporadic breast cancer by increasing local oestrogen concentration and that it is still a potential candidate for BRCA1.

Increased levels of nerve growth factor (NGF) protein and mRNA and reactive gliosis following kainic acid injection into the rat striatum

After excitotoxic lesion of the rat striatum, the time courses of local nerve growth factor (NGF) and NGF mRNA contents were investigated using a sensitive immunoassay (ELISA) and reverse transcription coupled to polymerase chain reaction (RT/PCR). To investigate a possible correlation of increased NGF expression and excitotoxin-induced reactive gliosis, striata were also analysed by immunohistochemistry with glial markers. We found elevated striatal NGF protein after lesion over the whole observation period. NGF mRNA showed a biphasic increase 10 h and 10 days after lesion, the latter co-inciding with an increased astrogliosis. These results indicate that NGF accumulation after excitotoxin-induced neurodegeneration is partly due to local reactive astrocytes.

Prolactin receptor gene expression in lymphoid cells

To understand the role of pituitary prolactin (PRL) and its receptor (PRL-R) in the growth and differentiation of lymphoid cells, PRL-R gene expression was analyzed in various lymphoid tissues and in a rat T lymphoma cell line, Nb2, which requires PRL for growth. The technique of reverse transcription coupled to polymerase chain reaction (RT-PCR) was used to detect the low abundance PRL-R transcripts. Within 30 min to 1 h, PRL stimulates a rapid but transient increase in PRL-R mRNA levels in Nb2 T cells. By 4 h, PRL-R mRNA returned to near basal levels and then gradually declined to a new steady-state level by 12 h. Significant increases in receptor RNA levels were observed in the presence of protein synthesis inhibitors, which suggests that PRL-R mRNA levels are under negative regulation. PRL-R gene expression was also demonstrated in normal mouse thymocytes, splenocytes, and in several lymphoid cell lines. The expression of the PRL-R gene in stimulated lymphoid cells provides additional evidence for the role of PRL as an immunomodulatory molecule.